L6 – Cellular respiration

Dr Agnieszka Adamczewska

Summer School 2015 Images from Wikimedia Commons

Major Concepts

1. Identify the three end-product options for glycolysis, and under what conditions these end-products form 2. State in words (not chemical formulae) the overall reaction of the glycolytic pathway, understand parts that are common and different 3. Understand how the overall balance sheet for glycolysis is obtained, and show the methods of reaction “coupling” that the cell uses 4. Say where the enzymes of the glycolytic pathway are to be found in eukaryotic cells and in prokaryotic cells 5. Explain what oxidation reduction reactions are and the special role of the coenzyme NAD + /NADH

Major Concepts

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7.

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9.

Give the names of, and recognise the equations for, the overall reactions of, the two major parts of the cellular respiration sequence - glycolysis and tricarboxylic acid (TCA) cycle.

Describe how a H + pumping mechanism is coupled to a proton-driven ATP synthase.

State how many ATP molecules are produced per glucose molecule in the glycolytic pathway and in the whole respiratory pathway.

Describe where in the respiratory pathway CO 2 released, and where O 2 is consumed.

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Macromolecules store energy

Energy is stored in carbon-carbon bonds e.g. glycogen or starch, fats and oils Get energy out of food through catabolic reactions Image from Campbell Biology 8e Australian Version © Pearson Education Inc.

Metabolism (L3)

• Metabolism: chemical reactions that occur within cells

Catabolism

energy – breaking down organic matter to release • Anabolism – using energy to produce cellular components Complex molecules Simple molecules Catabolism P i ADP Anabolism ATP

ATP: energy carrier

Made through metabolism of energy rich molecules.

- carbohydrates are converted into glucose - lipids are processed by β-oxidation Complex molecules Simple molecules Catabolism P i ADP ATP

ATP: Adenosine triphosphate

Hydrolysis ADP Adenosine diphosphate

Energy conversions in cells

Energy from macromolecules is released by and C-H bonds releases energy: cellular respiration.

Initial breakdown of macromolecules produces simple sugars, fatty acids, glycerol, and amino acids. Subsequent gradual oxidation of the fuel molecules by removal of electrons from C-C Energy conversions located in the cytosol 1. Glycolysis converts glucose to pyruvate 2. Fermentation to lactate and alcohol Energy conversions located in mitochondria in the presence of O 2 3.



-oxidation of lipids produces acetyl CoA 4. Citric acid cycle converts pyruvate to acetyl CoA and finally CO 2 5. Electron transport chain (NADH, FADH 2 > O 2 > H 2 O) drives proton pumps, proton gradient is coupled to synthesis of ATP

Energy conversion pathways Cellular respiration 1 3 CYTOSOL 2 MITOCHONDRION 5 4

Glycolysis

Pathway described in 1930's - a major biochemical triumph - involves a number of steps one glucose (C6) two pyruvate (2 x C3)

Glycolysis

Glucose (6C) 2 ATP 3 steps 2 ADP Fructose 1,6-bisphosphate (unstable) Cytosol NAD + G3P (3C) NADH 2 ADP 2 ATP 5 steps G3P (3C) glyceraldehyde 3 phosphate NAD + NADH 2 ADP 2 ATP Mitochondria image from Wikimedia Commons Pyruvate (3C) Pyruvate (3C)

Glycolysis Energy Conversions

Net yield 2 NADH 2 ATP

Glycolysis

“Splitting glucose” Glucose • from hydrolysis of polysaccharides (L2) • enters cell via facilitated diffusion - Glucose-Na + symport (L5) Cytosol – 10 enzymes Glucose (6C) + 2 ATP + 2 NAD + + 2 ADP + 2 Pi  2 Pyruvate (3C) + 4 ATP + 2 NADH Net yield: 2 ATP

NAD+ Nicotinamide Adenine Dinucleotide

Electron carrier (coenzyme )

NAD + reduced to NADH by transfer of H + from food

1. Glycolysis: Processing of glucose to pyruvate

a) Glucose (6C) is phosphorylated using 2 ATP and split into two molecules of glyceraldehyde 3-phosphate (3C) . Total 5 steps, consuming 2 ATP b) Oxidation in another 5 steps to 2 molecules of pyruvate (3C) and production of 4 ATP (net yield =2 ATP/glucose)

• •

Pyruvate can be converted in the absence of O fermentation to lactate (muscle tissue) 2 by alcoholic fermentation to ethanol (in yeast and bacteria) or by lactate In the presence of O 2 pyruvate enters mitochondria, is converted (decarboxylated) to a 2C compound acetyl CoA (a substrate for citric acid cycle) CO 2 , and NADH;

2. Fermentation Anaerobic conversion of pyruvate to alcohol or lactic acid

Alcohol fermentation

By yeast and many bacteria.

Used by humans for thousands of years in brewing, winemaking, and baking (CO 2 bubbles from baker’s yeast) REDUCTION

Lactic acid fermentation

By certain fungi and bacteria Used in dairy industry to make cheese and yogurt A product of muscle exercise, may enhance muscle performance (pain from K + ions) REDUCTION

Fermentation

No oxygen Glycolysis then fermentation 1 × Glucose Fungi, bacteria, animals  lactic acid 2 × Lactic acid Yeast, plants  ethanol + CO 2 1 × Glucose 2 × Ethanol 2ADP + 2P i 2ATP 2 × Pyruvate Glycolysis 2NAD + 2NADH + 2H + 2ADP + 2P i 2ATP 2 × Pyruvate Glycolysis 2NAD + 2NADH + 2H + 2 × CO 2

Food

Pyruvate

A key juncture in catabolism Chloroplast

Mitochondria (L4)

• Site of cellular respiration (energy production) • Double membrane: permeable outer membrane and impermeable, folded inner membrane (cristae) containing enzymes of respiration Outer membrane Inner membrane Crista Matrix Images from Wikimedia Commons

Mitochondria: number, shape, and subcellular location are highly variable

Fuel in, energy out

Glucose + oxygen  carbon dioxide + water + energy Cytosol NADH FADH 2 Krebs cycle Oxidative phosphorylation NADH Glycolysis Crista Matrix Mitochondria image from Wikimedia Commons ATP Intermembrane space ATP Mitochondria ATP

Intermediate reaction Pyruvate to Acetyl CoA

Outer mitochondrial membrane Inner mitochondrial membrane CoA Acetyl-CoA CoA CO 2 Matrix NADH + H + NAD + Pyruvate Pyruvate dehydrogenase complex Mitochondria image from Wikimedia Commons

Other fuel molecules

• Other carbohydrates (apart from glucose) • Fats and proteins can also be broken down and enter pathways 

-oxidation

Image from Campbell Biology 8e Australian Version © Pearson Education Inc.

3.

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-Oxidation of lipids

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-oxidation degrades long-chain fatty acids by 2C atoms at a time Last reaction splits off acetyl CoA (energy in C-C bond) and enters Citric acid cycle NADH, FADH2, energy (electron) carriers

8 steps, each catalysed by different enzyme

AcetylCoA (2C) 4. Krebs cycle

TCA cycle Citric acid cycle

Oxaloacetate (4C) 4C

NADH

4C 4C

FADH 2

Citrate (6C) 6C

Krebs cycle NADH NADH

5C

CO 2

4C

CO 2 ATP NAD + and FAD reduced to NADH and FADH 2 accept e- from intermediates CO 2 evolved

ATP

is formed

Chemical bonds to electrons

C 6 H 12 O 6 + 0O 2  6CO 2 + 0H 2 O + energy glucose 2 × NADH CO 2 6 × NADH 2 × FADH 2 Krebs cycle CO 2 Glycolysis Mitochondria image from Wikimedia Commons ATP ATP

Chemical bonds to electrons

1 glucose: Glycolysis  2 pyruvate + 2 ATP + 2 NADH 2 pyruvate  2 acetyl CoA + 2 NADH + 2 CO 2 TCA cycle: 2 acetyl CoA  6 NADH + 2 FADH 2 + 4 CO 2 + 2 ATP 1 glucose  4 ATP + 8 NADH + 2 FADH 2 + 6 CO 2

5. Electron transport chain (ETC)

(Proton-motive force i.e. the power in movement of protons) NADH and FADH 2 Inner membrane of mitochondria Four protein complexes of acceptors

Inner membrane

Oxygen needed Crista Matrix Mitochondria image from Wikimedia Commons

5. Electron transport chain

Start with NADH (or FADH 2 ) as primary electron donor

Intermembrane space

H + H + H +

H +

H + H + H +

H +

H + H + H + H +

C

Finish with O 2 as terminal electron acceptor

Q NADH e – H + NAD + FADH 2 H + FAD NADH dehydrogenase

bc 1

complex Mitochondrial matrix

H +

H +

H + H + H +

Inner mitochondrial membrane e – 2H + + 1/2O 2 H 2 O Cytochrome oxidase complex

Chain of redox reactions

Inner mitochondrial membrane Electrons move to higher redox potentials, towards oxygen with highest electron affinity Energy released is used to pump H + membrane space from matrix to inter NADH + H + Energy 2H + + NAD + released 2e Cyt b (Fe 3+ ) High energy Cyt b (Fe 2+ ) Cyt c (Fe 2+ ) Electrons “falling” from NADH to oxygen Cyt c (Fe 3+ ) Cyt a (Fe 3+ ) Cyt a (Fe 2+ ) H 2 O Low energy ½ O 2 + 2H +

Generating proton gradient

Start with NADH (or FADH 2 ) as primary electron donor

Intermembrane space

H + H + H +

H +

H + H + H +

H +

H + H + H + H +

C

Finish with O 2 as terminal electron acceptor

Q NADH e – H + NAD + FADH 2 H + FAD NADH dehydrogenase

bc 1

complex Mitochondrial matrix

H +

H +

H + H + H +

Inner mitochondrial membrane e – 2H + + 1/2O 2 H 2 O Cytochrome oxidase complex

Chemiosmosis couples the ETC to ATP synthesis!

OMM IMS IMM NADH + H + FADH 2 FAD NAD + H 2 O H + ½ O + OH 2 + 2H + H 2 O ADP + P i Electron transport chain ATP

ATP synthase

complexity of 1 o , 2 o , 3 o and 4 o structure embedded in inner membrane of mitochondria highly conserved in nature Image from http://www.rcsb.org/pdb/101/motm.do?momID=72 Intermembrane space Inner membrane Matrix

ATP synthase

H + ions flow down gradient and enter half channel in stator H + ions enter binding sites in rotor, changing shape of subunits so rotor spins Each H + ion makes one complete turn before being released to matrix H + binding Rotor H + H + H + H + Intermembrane space Inner membrane Catalytic knob Matrix Spinning of rotor causes internal rod to spin Turning rod activates catalytic sites in knob ADP + P i ATP Image from http://www.rcsb.org/pdb/101/motm.do?momID=72

Fuel in, energy out

Glucose + oxygen  carbon dioxide + water + energy Cytosol NADH FADH 2

2

Krebs cycle Oxidative phosphorylation NADH Glycolysis Crista Matrix 2x Mitochondria image from Wikimedia Commons ATP Intermembrane space 2x ATP Mitochondria ~34x ATP

Aerobic respiration = lots of energy

C 6 H 12 O 6 + 6 O 2  6 CO 2 + 6 H 2 O + ~38 ATP Where are these ATPs from?

Glycolysis = 2 ATP TCA = 2 ATP Oxidative phosphorylation (ETC + chemiosmosis) = ~34 ATP

Cristae increases surface area

• Infoldings of the inner mitochondrial membrane (cristae) greatly increase the number of electron transport chain proteins and ATP synthase proteins

Lack of oxygen?

Need oxygen to accept final electrons If no oxygen, complex IV keeps electrons     All protein complexes keep electrons No pumping of H + into intermembrane space No H + gradient No energy for ATP synthase No ATP made in ETC - ATP from glycolysis and TCA not enough Most cells cannot survive long without oxygen

Prokaryotic cell – no mitochondria

Still need ATP!

Plasma membrane

Capsule Nucleoid region Cell wall Image from Wikimedia Commons Pilus Ribosome Flagellum

Aerobic Bacteria do it too!

H+ ATP synthase complex ATP Enzymes embedded in bacterial cell membrane ADP + Pi Cell membrane NADH O 2 H 2 O NAD + Electron carrier H+

Anaerobic respiration

Prokaryotes living in anaerobic environment - no oxygen e.g. waterlogged soils, intestines Nitrate (NO 3 ) or sulfate (SO 4 2 ) are the terminal electron acceptors Products include CO 2 , inorganic substance, ATP e.g. C 6 H 12 O 6 + 12 KNO 3 (potassium nitrate)  6 CO 2 + 6 H 2 O + 12 KNO 3 (potassium nitrite) + ATP

Summary

Cytoplasm and mitochondria are sites of cellular respiration Glucose + oxygen  carbon dioxide + water + energy Aerobic respiration has four stages For one glucose molecule: Glycolysis = 2 ATP, TCA = 2 ATP, Oxidative phosphorylation (ETC, chemiosmosis) = ~34 ATP Chemiosmosis - electrons in ETC used to pump protons into inter-membrane space, this establishes a proton gradient across inner membrane, protons accumulate, lowers pH Redox reactions integral to ETC Fermentation and anaerobic respiration in absence of O 2

Aerobic respiration

Stage Where Main starting materials Main end products Glycolysis Cytoplasm forming Acetyl CoA Matrix of mitochondria Citric acid cycle Matrix of mitochondria Oxidative phosphorylation Inner membrane mitochondria Glucose Pyruvate, ATP , NADH Pyruvate Acetyl CoA, H 2 O Acetyl CoA, CO 2 , NADH CO 2 , NADH, FADH 2 , ATP O 2 , NADH, FADH 2 ATP , H 2 O

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Read:

• Knox B, et al. (2010)

Biology: an Australian perspective.

- Chapter 6 Harvesting energy